Forward genetics + a bit of common tools II

Question 1

What is the target molecule of southern blot?

Answer 1

DNA

Question 2

What is the target molecule of northern blot?

Answer 2

RNA

Question 3

What is the target molecule of western blot?

Answer 3

Proteins

Question 4

What is forward genetics?

Answer 4

The process of starting out with a phenotype (often unlike the wildtype or most common trait) and find the responsible gene(s) for the phenotype.

Question 5

What is reverse genetics?

Answer 5

The process of starting out with a known gene, and induce changes (permanent or non-permanent) to this gene to figure out what phenotype(s) it causes.

Question 6

List the three main functional effects of a loss of function (LOF) mutation.

Answer 6

Null/amorphic: complete inhibition of expression of a gene, could be because of a deletion.

Leaky/hypomorphic: weaker expression than normal, but not completely deleted. Could be because of a mutation in the promoter, RNA Pol cannot bind as efficiently for ex.

Dominant negative: If the organism is heterozygous for the mutation, it can be dominant negative. The product of the mutated allele may interfere with the function of the WT allele, for example by that the non-functional protein competes with the normal one, making it look like there is an overall weaker expression of the gene.

Question 7

List the main functional effects of a gain of function (GOF) mutation.

Answer 7

Hypermorphic: expression of more gene product than normal, but no change in the actual function of the protein.

Neomorphic: the mutant gains a new gene function that is different from the WT. Often dominant.

Question 8

What is the difference between incomplete dominance and codominance? Give one example

Answer 8

Incomplete dominance: color of flower petals, where homozygotes are either red or white but heterzygotes are pink = an intermediate between the alleles.

Codominance: rather than being an intermediate, the heterozygotes show both traits. For example blood groups.

Question 9

What is pleiotropy?

Answer 9

The phenomenon where one gene governs multiple phenotypes.

Question 10

What is a conditional mutant?

Answer 10

A mutant that only exhibits the mutant phenotype under specific conditions. E.g temperature.

Question 11

Give two examples on cases where the allele can show variable phenotypes.

Answer 11

Incomplete penetrance: when the phenotype is not always prevalent even though the genotype is present, and also dominant. This leads to that the trait “skips” generations and shows up randomly in individuals. Autosomal dominant.

Variable expressitivity: the intensity/level of the phenotype varies among individuals. Autosomal dominant.

Question 12

What two main things does the randomness of chromosomal inheritance depend on?

Answer 12

Independent assortment: In Metaphase I of Meiosis I, the replicated, homologous chromosomes line up in the center of the cell in a random manner. This means that the frequency of maternal and paternal chromosomes that goes into each daughter cell is random - it’s independent.

Homologous recombination: In prophase I (just before metaphase) the replicated, homologous chromosomes (maternal and paternal) align and perform a cross-over. Because of this, the inheritance of separate genes is not dependent on other genes.

Question 13

Give three examples on how mutations can be induced.

Answer 13

Chemical mutagenesis: EMS = ethyl methanesulfonate. This adds ethyl an ethyl group to guanine residues, making it base pair to thymine instead of cytosine –> inducing a GC to AT mutation.

Irridation: UV light can produce a cross linking between adjacent Thymine residues, which leads to that the residues are interpreted as one instead of two by the replication fork –> -1 frameshift mutation!

Insertion: e.g transposons.

Question 14

Which generation do you mutagenize?

Answer 14

Parental generation, P

Question 15

Why are only dominant mutations visible in the F1 generation?

Answer 15

You usually mutagenize the sperm cells and then cross the male to a WT female. Since the only one of the chromosomal sets have the mutation, it has to be dominant in order to be visible in the F1 generation.

Question 16

What is an F2 screen? What kinds of mutations can be visible in the F2 generation? What kind of organisms is best fitted for this screen?

Answer 16

Self-fertilizing organisms is best for an F2 screen, since recessive mutations can be visualized in the F2 generations if the organism is self-fertilizing.

Question 17

Describe the process of an F2 screen.

Answer 17

1. Mutagenize the germ cells in the P generation. Only random germ cells will carry the mutation.
2. Cross the P generation for the F1 gen, the F1 individuals will carry eggs and sperm cells with the mutation.
3. Self-fertilize the F1 generation –> around 25% of the F2 offspring will be homozygous for the recessive mutation and show a visible phenotype.

Question 18

What is the reason a recessive mutation cannot be visualized in the F2 generation in non self fertilizing organisms?

Answer 18

Because in the F1 generation, each mutagenized sperm cell is unique. This means that the F1 individuals first have to be crossed with each other in order for the F2 individuals to carry the same mutated alleles. Thereafter, the F2 can be crossed for the mutation to be visualized in the F3 generation.

Question 19

What is an F3 screen? What kinds of mutations can be visible in the F3 generation? What kind of organisms is best fitted for this screen?

Answer 19

An F3 screen is best fitted for when you want to visualize recessive mutations in non-self fertilizing organisms.

Question 20

Describe the process of an F3 screen.

Answer 20

1. Mutagenize sperm cells of P generation, mate the mutated males with WT females –> F1 offspring.
2. Isolate the F1 progeny that was the offspring from each mutated male mated with WT female –> each F1 individual has a unique mutation. Each F1 individual is crossed individually to a WT to create F2 families.
3. Each F2 family carries one type of mutation. The individuals in each family is crossed with each other to create F3 progeny. Some of the F3 individuals should show phenotypes of a recessive mutation.

Question 21

What is a complementation group?

Answer 21

Complementation group = groups of mutations that fail to complement each other, i.e they are likely located on the same gene.

Mutations from different complementation groups can complement each other and will produce a WT phenotype when crossed with each other, since the homologous gene of the other individual is wiltype (not mutated).

Question 22

Mutagenesis often produces many mutations in the genome. How can you reduce the number of different mutations in a mutated strain?

Answer 22

By outcrossing the strain with a WT strain –> will reduce background noise.

Question 23

Whole genome sequencing is a way to detect in which chromosome/gene an induced mutation is located, but it’s time-consuming and expensive to sequence the whole genome. How can you narrow down the location of the mutation before sequencing?

Answer 23

1. Mapping by recombination: cross the mutant with another strain carrying a genetic marker, e.g SNP. The lower the frequency of offspring carrying both the marker and the mutation, the closer they are on the chromosome, because it requires recombination. When narrowed down –> complementation.
2. Complementation. To find out only how many different genes the mutations are on –> group into complementation groups.
3. Sequence.

Question 24

What is the definition of an epistatic gene?

Answer 24

An allele of a gene that masks the phenotype of another gene is epistatic to that gene.

Question 25

Give an example of how a gene can be epistatic over another.

Answer 25

Different alleles of the same gene can produce different hair colors, but the presence of an allele on another gene that causes baldness will mask the other gene for hair color, regardless of which allele it carries.

Question 26

What kind of probes are used in western blot?

Answer 26

Labeled antibodies - primary or secondary

Question 27

What kinds of probes are used in southern and northern blot?

Answer 27

Nucleic acid probes that are labeled

Question 28

What are the three steps of purification of DNA, RNA and proteins?

Answer 28

1. Lysis of cells
2. Enrich for the nucleic acid/protein you want. DNA and RNA are more homogenous than proteins and therefore easier to enrich for. For proteins, you have to adapt the method for the specific protein of interest.
3. Increase purity and remove contaminants. DNase and RNase can be added when purifying proteins, and nucleic acids can be precipitated.